Many diseases are caused or characterised by an imbalance in the number of chromosomes (aneuploidy) or an imbalance in the number of chromosomal segments (partial aneuploidy) in cells of an individual compared with the normal number of chromosomes or chromosomal segments for the species. The human diploid genome has 23 pairs of chromosomes; paired chromosomes 1 to 22 and the sex chromosomes XX or XY. The terms monosomy and trisomy refer to a missing or extra chromosome, while partial monosomy and partial trisomy refer to an imbalance of genetic material caused by loss or gain respectively of part of a chromosome. Aneuploidy and partial aneuploidy in an individual's genome are associated with congenital disorders such as Down's syndrome (trisomy of human chromosome 21) and Turner syndrome (monosomy or partial monosomy of the sex chromosome). Aneuploidy and partial aneuploidy may also arise through somatic mutation in adult tissues. For example, many cancer cells exhibit chromosomal fragility leading to translocations of chromosomal fragments and aneuploidy of tumour cells.
Methods have been developed for diagnosing diseases associated with chromosomal defects. Traditional methods of karyotyping included obtaining a tissue sample, staining the chromosomes and examining them under a light microscope. Schröck et al. (Science 273(5274):494-497 1996) described multicolour spectral karyotyping, using fluorescence in situ hybridisation (FISH) to simultaneously visualise all human chromosomes in different colours. Fluorescently labelled probes were made for each chromosome by labelling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally distinct fluorophores, a combinatorial labelling method was used to generate the required number of different emission spectra. Spectral differences generated by combinatorial labelling were captured and analysed using an interferometer attached to a fluorescence microscope. Image processing software then assigned a colour to each spectrally different combination, allowing the visualisation of the individually coloured chromosomes.
Comparative genomic hybridisation (CGH) involves the isolation of DNA from the two sources to be compared, most commonly a test and reference source, independent labelling of each DNA sample with fluorophores of different colours (usually red and green), denaturation of the DNA so that it is single stranded, and the hybridisation of the two resultant samples in a 1:1 ratio to a normal metaphase spread of chromosomes, to which the labelled DNA samples will bind at their locus of origin. Using a fluorescence microscope and computer software, the differentially coloured fluorescent signals are then compared along the length of each chromosome for identification of chromosomal differences between the two sources. A higher intensity of the test sample colour in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample colour indicates the loss of material in the test sample in that specific region. A neutral colour (yellow when the fluorophore labels are red and green) indicates no difference between the two samples in that location. CGH was described by Kallioniemi et al., Science 258(5083):818-21 1992 and Pinkel et al., Nat Genet. 20(2):207-11 1998.
More recently, digital or virtual karyotyping methods have been developed to quantify copy number on a genomic scale (Wang et al., PNAS 99(25):16156-16161 2002). Digital karyotyping allows differences in copy number to be detected at higher resolution compared with conventional karyotyping or chromosome-based CGH. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Tags of 21 bp each can be obtained from specific locations in the genome and generally contain sufficient information to uniquely identify the genomic loci from which they were derived. Tags can thus be matched to precise chromosomal locations and tag densities can be evaluated over moving windows to detect abnormalities in DNA sequence content. Methods of matching the sequence tags to their chromosomal locations include high throughput sequencing, use of array-comparative genomic hybridisation and SNP arrays.
Arrays are composed of hundreds to millions of probes which are complementary to a region of interest in the genome. DNA from the test sample is fragmented, labelled, and hybridised to the array. The hybridisation signal intensities for each probe are quantified for each position on the array. Knowing the address of each probe on the array and the address of each probe in the genome, an algorithm is used to line up the probes in chromosomal order and reconstruct the genome in silico. The resolution of digital karyotyping depends on the density of probes on the array.
One area where high precision analysis is required is in non-invasive prenatal karyotyping. Pregnant mothers carry cell-free circulating DNA in their blood, of which 4-30% is derived from the foetus. It is possible to determine the karyotype of the foetus by determining the abundance of cell free DNA originating from each chromosome. For example, if the cell free DNA consists of 95% maternal and 5% foetal DNA, and if the foetus has trisomy of chromosome 21 (Down's syndrome) then the total amount of cell free DNA from chromosome 21 should exceed that of any other genomic region of the same size by 2.5%. Observing a chromosomal aneuploidy in the foetal DNA requires a very precise measurement to detect such slight imbalances in the relative quantities of different chromosomes. The difficulty is compounded by a need to work with relatively small samples in order to provide a method that is convenient and acceptable for patients and clinicians.
Analysis of specific targets from single or a few DNA molecules has traditionally been a technical challenge. Methods to copy DNA are typically required to achieve sufficient signal for downstream analysis procedures. Analysis methods such as DNA sequencing, gel electrophoresis, and DNA microarrays typically require a signal amplification of the DNA in the sample provided. The most common amplification method to amplify specific DNA targets is PCR, which can provide millions (or billions) of copies of specific targets from a DNA sample. However, when it is desired to amplify many regions of a genomic sample for analysis, amplification artefacts can arise as a result of performing multiple different amplifications together in the same reaction mixture. Also, an amplification step can result in loss of information regarding relative quantities of sequences in the sample, since the original difference in relative quantity may be tiny compared with the absolute magnitude of the amplified nucleic acid products, and since different sequences may be amplified with different efficiencies.